Conflict of interest: No conflict of interest has been declared. Nonstandard abbreviations used: free cholesterol (FC); acylcoenzyme A: cholesterol acyltransferase (ACAT); cholesteryl ester (CE); Niemann-Pick C protein 1 (npc1); endoplasmic reticulum (ER); phosphatidylcholine (PC); CTP: phosphocholine cytidylyltransferase (CT). groups of membrane phospholipids is critical for the formation of liquid-ordered rafts (10). Thus, cholesterol depletion causes marked disruption of these rafts. However, when the FC/phospholipid ratio rises above a physiological level, the liquid-ordered rafts may become too rigid, and the liquid-crystalline domains may begin to lose their fluidity. These events adversely affect certain integral membrane proteins that require conformational freedom for proper function and that can be inhibited by a high FC/phospholipid ratio (14). Such proteins include plasma membrane constituents like Na+-K+ ATPase, adenylate cyclase, alkaline phosphatase, rhodopsin, and transporters for glucose, organic anions, and thymidine. Similar observations have been made with proteins residing in internal membranes, such as the Na+-Ca2+ transporter in the sarcoplasmic reticulum of cardiac muscle, the ATPADP transporter in the inner mitochondrial membrane, and UDP-glucuronyltransferase in liver microsomes (14). Interestingly, inhibition of ACAT activity in Chinese hamster ovary cells transfected with amyloid precursor protein blocks the generation of amyloid β-peptide (15). Although the mechanism of this effect is not yet known, one possibility is that the conformation of the amyloid precursor or the β-peptide–generating proteases is altered by an increase in the local FC/phospholipid ratio (Simons and Ehehalt, this series, ref. 11). High FC levels might therefore be proposed to kill cells in part by inhibiting one or more integral membrane proteins whose function is blocked or altered under conditions of high membrane rigidity. This and several other models for FC-induced cell death, discussed below, are summarized in Table 1. Excess membrane cholesterol may also disrupt the function of signaling proteins that reside in membrane domains. For example, when human neutrophils with a normal plasma membrane cholesterol content are stimulated to migrate by exposure to chemokines, the actin-signaling protein Rac is recruited to detergent-sensitive non-raft membrane domains in leading lamella. However, when the plasma membrane of these cells are overloaded with cholesterol, Rac is recruited to the entire circumference of the plasma membrane, lamellar extension is nonvectorial, and neutrophil migration does not occur (LM Pierini and FR Maxfield, personal communication). One interpretation of these data is that excess plasma membrane cholesterol disrupts the function of certain signaling molecules that normally reside in non-raft domains. Experiments in vitro with model membranes suggest that, in membranes already enriched in sphingolipids (like those that exist in several types of epithelial cells), increasing the FC concentration even modestly above the physiological concentration can actually suppress the formation of membrane domains (16). Other mechanisms of cellular toxicity associated with FC accumulation include intracellular cholesterol crystallization, oxysterol formation (Björkhem, this series, ref. 8), and triggering of apoptotic signaling pathways (9). Needle-shaped cholesterol crystals form when the FC/phospholipid ratio reaches a very high level. Although typically seen in extracellular regions of advanced atherosclerotic lesions, intracellular cholesterol crystals have been observed both in cultured macrophages overloaded …